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To have information on the proteolytic activity of convertases and exo-peptidases on human salivary proteins, this study investigated the relative amounts of the truncated proteoforms in the saliva of preterm newborns and compared them with the relative amounts measured in saliva of at-term newborns, of babies (0-10 years old) and of adults. Results indicated that convertase(s), acting on acidic proline-rich proteins and histatin 3, and carboxypeptidase(s) acting on acidic proline-rich proteins, P-C peptide, histatin 6 and statherin were many folds more active in preterm newborns than in the other groups. Conversely, the aminopeptidase responsible for the removal of the N-terminal Asp residue of statherin was not active in preterm newborns, becoming active only several months after the normal term of delivery. The high activity of convertases determined in preterm newborns suggests that it is required for the molecular events connected to the fetus development, and encourages further studies devoted to the characterization of their specific substrates., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Gingival Crevicular Fluid (GCF), a plasma-derived exudate present in the gingival crevice was collected from deciduous, exfoliating and permanent teeth from 20 children (60 samples) with the aim to characterize and quantify by a mass spectrometry based top-down proteomic approach, the peptide/proteins in the fluid and verify possible variations occurring during the exfoliating process. The results obtained confirmed the presence in GCF of α-Defensins 1-4, Thymosin β4 and Thymosin β10, as described in previous works and revealed the presence of other interesting peptides never described before in GCF such as specific fragments of α-1-antitrypsin, α-1-antichymotrypsin; fragments of Thymosin β4 and Thymosin β10; Fibrinopeptide A and its fragments and Fibrinopeptide B; S100A8 and S100A9, LVV Hemorphin-7 (hemoglobin chain β fragment), as well as some other peptides deriving from α and β subunits of hemoglobin. Statistical analysis evidenced different levels in 5 proteins/peptides in the three groups. Our study demonstrate that an in-depth analysis of a biological fluid like GCF, present in small amount, can provide useful information for the understanding of different biological processes like teeth eruption. Data are available via ProteomeXchange with identifier PXD016010 and PXD016049. SIGNIFICANCE: GCF due to his site-specific nature has a great potential in containing factors that are specific for action at a given site and might have diagnostic value to detect qualitative and quantitative variations of proteins/peptides composition linked to physiological or pathological conditions., Competing Interests: Declaration of Competing Interest The authors declare no competing financial interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The multifactorial nature of Late Onset Alzheimer's Disease (LOAD), the AD form of major relevance on epidemiological and social aspects, has driven the original investigation by LC-MS and top-down proteomics approach of the protein repertoire of the brain tissue of TgCRND8 model mice fed with a diet deficient in B vitamins. The analysis of the acid-soluble fraction of brain tissue homogenates identified a list of proteins and peptides, proteoforms and PTMs. In order to disclose possible modulations, their relative quantification in wild type and AD model mice under both B vitamin deficient and control diets was performed. The levels of metallothionein III, guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2 and brain acid soluble protein 1 showed statistically significant alterations depending on genotype, diet or both effects, respectively. Particularly, metallothionein III exhibited increased levels in TgCRND8 mice under B vitamin deficient diet with respect to wild type mice under both diets. Brain acid soluble protein 1 showed the opposite, revealing decreased levels in all diet groups of AD model mice with respect to wild type mice in control diet. Lower levels of brain acid soluble protein 1 were also observed in wild type mice under deficiency of B vitamins. These results, besides contributing to increase the knowledge of AD at molecular level, give new suggestions for deeply investigating metallothionein III and brain acid soluble protein 1 in AD., (Copyright © 2019. Published by Elsevier B.V.)

PET scan analysis demonstrated the early reduction of cerebral glucose metabolism in Alzheimer disease (AD) patients that can make neurons vulnerable to damage via the alteration of the hexosamine biosynthetic pathway (HBP). Defective HBP leads to flawed protein O-GlcNAcylation coupled, by a mutual inverse relationship, with increased protein phosphorylation on Ser/Thr residues. Altered O-GlcNAcylation of Tau and APP have been reported in AD and is closely related with pathology onset and progression. In addition, type 2 diabetes patients show an altered O-GlcNAcylation/phosphorylation that might represent a link between metabolic defects and AD progression. Our study aimed to decipher the specific protein targets of altered O-GlcNAcylation in brain of 12-month-old 3×Tg-AD mice compared with age-matched non-Tg mice. Hence, we analysed the global O-GlcNAc levels, the levels and activity of OGT and OGA, the enzymes controlling its cycling and protein specific O-GlcNAc levels using a bi-dimensional electrophoresis (2DE) approach. Our data demonstrate the alteration of OGT and OGA activation coupled with the decrease of total O-GlcNAcylation levels. Data from proteomics analysis led to the identification of several proteins with reduced O-GlcNAcylation levels, which belong to key pathways involved in the progression of AD such as neuronal structure, protein degradation and glucose metabolism. In parallel, we analysed the O-GlcNAcylation/phosphorylation ratio of IRS1 and AKT, whose alterations may contribute to insulin resistance and reduced glucose uptake. Our findings may contribute to better understand the role of altered protein O-GlcNAcylation profile in AD, by possibly identifying novel mechanisms of disease progression related to glucose hypometabolism., (Copyright © 2018. Published by Elsevier B.V.)

Multiple sclerosis is a chronic disease of the central nervous system characterized by inflammation, demyelination and neurodegeneration which is of undetermined origin. To date a single diagnostic test of multiple sclerosis does not exists and novel biomarkers are demanded for a more accurate and early diagnosis. In this study, we performed the quantitative analysis of 119 salivary peptides/proteins from 49 multiple sclerosis patients and 54 healthy controls by a mass spectrometry-based top-down proteomic approach. Statistical analysis evidenced different levels on 23 proteins: 8 proteins showed lower levels in multiple sclerosis patients with respect to controls and they were mono- and di-oxidized cystatin SN, mono- and di-oxidized cystatin S1, mono-oxidized cystatin SA and mono-phosphorylated statherin. 15 proteins showed higher levels in multiple sclerosis patients with respect to controls and they were antileukoproteinase, two proteoforms of Prolactin-Inducible Protein, P-C peptide (Fr.1-14, Fr. 26-44, and Fr. 36-44), SV1 fragment of statherin, cystatin SN Des 1-4 , cystatin SN P 11  → L variant, and cystatin A T 96  → M variant. The differences observed between the salivary proteomic profile of patients suffering from multiple sclerosis and healthy subjects is consistent with the inflammatory condition and altered immune response typical of the pathology. Data are available via ProteomeXchange with identifier PXD009440., Significance: To date a single diagnostic test of multiple sclerosis does not exist, and diagnosis is based on multiple tests which mainly include the analysis of cerebrospinal fluid. However, the need for lumbar puncture makes the analysis of cerebrospinal fluid impractical for monitoring disease activity and response to treatment. The possible use of saliva as a diagnostic fluid for oral and systemic diseases has been largely investigated, but only marginally in multiple sclerosis compared to other body fluids. Our study demonstrates that the salivary proteome of multiple sclerosis patients differs considerably compared to that of sex and age matched healthy individuals and suggests that some differences might be associated with the different disease-modifying therapy used to treat multiple sclerosis patients., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The acid-insoluble salivary proteome obtained by addition of TFA to whole human saliva from adults, preterm and at-term newborns has been analysed by 2-DE in order to evidence differences among the three groups, and integrate data previously obtained on the acid-soluble fraction. 2-DE spots differentially expressed among the three groups were submitted to in-gel tryptic digestion and the peptide mixtures analysed by high resolution HPLC–ESI–MS/MS. By this strategy, we identified 3 over-expressed proteins in at-term newborns with respect to preterm newborns and adults (BPI fold-containing family A member 1, annexin A1, and keratin type 1 cytoskeletal 13), and several over-expressed proteins in adults (fatty acid-binding protein, S100 A6, S100 A7, S100 A9, prolactin-inducible protein, Ig kappa chain, cystatin SN, cystatin S/SA and α-amylase 1). Four spots, already detected but not characterized by other authors in human saliva 2-DE, were attributed to different protein species of S100 A9 (long-type and long-type monophosphorylated, short-type and short-type monophosphorylated) by MS/MS analysis of tryptic peptides and sequential staining of 2-DE gels with Pro-Q Diamond, for specific detection of phosphoproteins, and total protein SYPRO Ruby stain., Significance: Differential protein expression analysis of the acid insoluble fraction of saliva from preterm, at-term newborns and adults has been performed in this study by coupling 2-DE analysis and high-resolution tandem mass spectrometry in order to complete the information previously obtained by top-down LC–MS only on the acid-soluble proteome. Several proteins identified in the acid insoluble fraction of both preterm newborn and adult saliva are not of glandular origin, being only prolactin-inducible protein, salivary cystatins, α-amylase and polymeric immunoglobulin receptor exclusive of salivary glands. Three proteins resulted increased in at-term newborns with respect to preterm newborns and adults: BPI fold-containing family A member 1, two proteoforms of annexin A1 and keratin type 1 cytoskeletal 13, while several proteins were significantly increased in adults.

Unlabelled: The most heterogeneous family of human salivary proteins is represented by proline-rich proteins (PRPs) divided in acidic, basic, and basic glycosylated (aPRPs, bPRPs, gPRPs). They are encoded by six genes, clustered on chromosome 12p13.2: PRH1-2 encode aPRPs, PRB1-4 encode bPRPs and gPRPs. Each gene exists in different allelic forms: two for PRH2, three for PRH1, PRB2, and PRB4, four for PRB1, and PRB3. During granule maturation, PRP proproteins undergo proteolysis by the action of convertases and carboxypeptidases. Differently from bPRPs, proteolysis of aPRPs is not complete, and, besides fragments, entire protein species are also secreted. Maturation process generates ten aPRPs (PRP-1, PRP-2, PIF-s, Db-s, Pa, PRP-3, PRP-4, PIF-f, Db-f, P-C), and at least 18 bPRPs (II-2, P-E, IB-6, Ps-1, Ps-2, IB-1, P-J, IB-8a, P-F, P-H, P-D, II-1, protein glycosylated A, CD-IIg, and Gl1-4). In addition, single nucleotide and length polymorphisms, and differentially spliced transcripts originate several natural variants. Phosphorylation, N-pyroglutaminylation, dimerization, and N-/O-glycosylation also occur during maturation, enlarging the number of protein species, further increased by proteolytic events governed by carboxy- and endo-peptidases during and after secretion, and giving rise to a huge number of small peptides. The PRP functional role is still poorly understood., Significance: The high polymorphism of PRPs gives an important contribution to the high heterogeneity and inter-individual variability of the human salivary proteome. The products of six genes clustered on chromosome 12p13.2 comprise a mixture of entire, truncated, phosphorylated, glycosylated and dimerized protein/peptide species, sharing large part of their sequences, and possibly involved in different biological activities. Whatever the role of PRP species is, it should be crucial, given that PRPs are the most conserved oral salivary proteins among mammals., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The goal of this study was to develop an optimized drug delivery carrier for oral mucosa applications able to release in situ bioactive molecules by using biopolymeric materials. Among them chitosan and poly(lactic-co-glycolic acid) (PLGA) have gained considerable attention as biocompatible carriers able to improve the delivery of active agents. The formulation of such vehicles in the form of nanoparticles (NPs) could permit to exploit the peculiar properties of nanomaterials in order to enhance the efficacy of active agents. Chitosan (CS) and PLGA chlorexidine dihydrochloride (CHX)-loaded NPs were synthesized by ionotropic gelation and osmosis based methodology respectively. In order to facilitate NPs adhesion on human dental surfaces, two different strategies were employed: PLGA particles with an external shell of CS to produce a positive surface charge allowing CHX loaded PLGA NPs to interact with the negative charged dental surfaces, while CS particles were functionalized with peptidomimetic derivative glutathione (GSH). The morphology was investigated by scanning electron microscopy. A sustained release profile of CHX from CS NPs was achieved. CS-based NPs adhered on human tooth surfaces in a simulated brushing and rinsing process and their in vitro toxicity evaluation on Human Gingival Fibroblasts (HGFs) was between 20 and 60% in all experimental conditions. Thanks to their adhesion properties and low cytotoxicity, the synthesized CS-based formulations may be efficiently exploited for therapy purposes or to enhance in vivo dental care (i.e. preparation of toothpastes or other cosmetics for daily oral care)., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Wilson's disease is a rare inherited disorder of copper metabolism, manifesting hepatic, neurological and psychiatric symptoms. Early diagnosis is often unfeasible and a unique diagnostic test is currently inapplicable. We performed the qualitative/quantitative characterization of the salivary proteome/peptidome of 32 Wilson's disease patients by an integrated top-down/bottom-up approach. Patients exhibited significant higher levels of S100A9 and S100A8 proteoforms, and their oxidized forms with respect to controls. Oxidation occurred on methionine and tryptophan residues, and on the unique cysteine residue, in position 42 in S100A8, and 3 in S100A9, that generated glutathionylated, cysteinylated, sulfinic, sulfonic, and disulfide dimeric forms. Wilson's disease patient saliva showed high levels of two new fragments of the polymeric immunoglobulin receptor, and of α-defensins 2 and 4. Overall, the salivary proteome of Wilson's disease patients reflected oxidative stress and inflammatory conditions characteristic of the pathology, highlighting differences that could be useful clues of disease exacerbation., (Copyright © 2015 Elsevier B.V. All rights reserved.)

A new photoactivatable trifunctional cross-linker, cBED (cadaverine-2-[6-(biotinamido)-2-(p-azidobenzamido) hexanoamido]ethyl-1,3'-dithiopropionate), was synthesized by chemical conversion of sulfo-SBED (sulfosuccinimidyl-2-[6-(biotinamido)-2-(p-azidobenzamido) hexanoamido]ethyl-1,3'-dithiopropionate) with cadaverine. This cross-linker was purified by reversed-phase high-performance liquid chromatography (RP-HPLC) and characterized using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) analysis. cBED is based on sulfo-SBED that has a photoactivatable azido group, a cleavable disulfide bond for label transfer methods, and a biotin moiety for highly sensitive biotin/avidin detection. By ultraviolet (UV) light, the azido group is converted to a reactive nitrene, transforming transient bindings of interacting structures to covalent bonds. In contrast to the sulfo-N-hydroxysuccinimide (sulfo-NHS) moiety of sulfo-SBED, which attaches quite unspecifically to amino groups, cBED includes a cadaverine moiety that can be attached by transglutaminase more specifically to certain glutamine residues. For instance, thymosin β4 can be labeled with cBED using tissue transglutaminase. By high-resolution HPLC/ESI-MS (electrospray ionization-mass spectrometry) and tandem MS (MS/MS) of the trypsin digest, it was established that glutamine residues at positions 23 and 36 were labeled, whereas Q39 showed no reactivity. The covalent binding of cBED to thymosin β4 did not influence its G-actin sequestering activity, and the complex could be used to identify new interaction partners. Therefore, cBED can be used to better understand the multifunctional role of thymosin β4 as well as of other proteins and peptides., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The analysis of whole saliva of 32 subjects with diagnosis of schizophrenia (SZ), 17 with diagnosis of bipolar disorder (BD), and 31 healthy subjects divided in non-smokers (HN; n=19) and smokers (HS; n=12) using an HPLC-ESI-MS top-down platform is reported in this study. Both SZ and BD revealed more than 10 fold mean increase of α-defensins 1-4, S100A12, cystatin A and S-derivatives of cystatin B levels with respect to the HN and HS control groups. No differences of protein levels were observed between SZ and BD groups and between HN and HS groups. Moreover, the correlation coefficients among the different proteins were significantly better in BD group than in SZ group., Biological Significance: This study on whole saliva confirms a schizophrenia-associated dysregulation of immune pathway of peripheral white blood cells and suggests that the dysregulation of BD group could involve the activation of more specific cell type than that of SZ group., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Objective: Thymosin beta 4 (Tβ4) is a ubiquitous peptide that plays pivotal roles in the cytoskeletal system and in cell differentiation. Recently, a role for Tβ4 has been proposed in experimental and human carcinogenesis, including gastrointestinal cancer. This study was aimed at evaluating the relationship between Tβ4 immunoreactivity and the initial steps of carcinogenesis., Methods: In total, 60 intestinal biopsies, including 10 hyperplastic polyps, 10 sessile serrated adenomas/polyps, 15 colorectal adenomas with low-grade dysplasia, 15 adenomas with high-grade dysplasia, 15 adenocarcinomas and 10 samples of normal colon mucosa, were analyzed for Tβ4 expression by immunohistochemistry., Results: Weak cytoplasmic reactivity for Tβ4 was detected in the normal colon mucosa. No reactivity for Tβ4 was found in hyperplastic and sessile serrated polyps/adenomas. Tβ4 expression was observed in 10/15 colorectal adenocarcinomas. In adenomas with low-grade dysplasia, Tβ4 immunoreactivity was mainly detected in dysplastic glands but was absent in hyperplastic glands. Tβ4 immunoreactivity was characterized by spot-like perinuclear staining. In high-grade dysplastic polyps, immunostaining for Tβ4 appeared diffuse throughout the entire cytoplasm of dysplastic cells. Spot-like perinuclear reactivity was detected in adenocarcinoma tumor cells., Conclusions: Our study shows for the first time that Tβ4 is expressed during different steps of colon carcinogenesis. The shift of Tβ4 immunolocalization from low-grade to high-grade dysplastic glands suggests a role for Tβ4 in colorectal carcinogenesis. However, the real meaning of Tβ4 reactivity in dysplastic intestinal epithelium remains unknown.

A CE-tandem MS method was optimised and validated for selective and specific determination of LVV- and VV-hemorphin-7 peptides in cerebrospinal fluid. These two small peptides originate from haemoglobin beta chains. They possess relevant biological activity and recently a potential biomarker role in posterior cranial fossa paediatric brain tumour disease was evidenced. The separation was optimised using formic acid as background electrolyte and a water/methanol mixture, containing 0.1% (v/v) formic acid, as sheath liquid. The two peptides, differing in only one amino acid of the sequence at the N-terminal side were baseline separated in less than 15 min. The method allowed a very reduced and rapid sample pretreatment and was successfully applied to hemorphins determination in patient samples without matrix interferences. The method successfully passed bioanalytical validation showing linearity, accuracy and precision data on cerebrospinal fluid matrix within the acceptable values. The analysis of cerebrospinal fluid of patients affected by different posterior cranial fossa tumour forms confirmed our previous findings showing the absence of hemorphins in the pre-surgical cerebrospinal fluid and their presence in the post-ones and controls. The present method saves costs and time due to capillary electrophoresis miniaturisation and to the absence of chromatographic column and gradient elution and allows numerous injections per sample starting from few microlitres of cerebrospinal fluid., (Copyright © 2012 Elsevier B.V. All rights reserved.)

An HPLC-ESI-MS analysis of adult human whole saliva evidenced three protein masses (M average 11,487±2, 11,301±2 and 22,362±3Da) eluting in the 32.5-35.0min range. Treatment in reducing conditions allowed establishing that they are S-derivatives of N-terminal acetylated cystatin B, namely its S-glutathionyl, S-cysteinyl and S-S dimer. The identification was confirmed by high resolution HPLC-ESI-MS-MS experiments on the intact naturally occurring proteins and their tryptic digests. S-unmodified cystatin B is rarely detectable in whole saliva of healthy adults (5 subjects out of 65) and its percentage does not overcome approximately 20% of total cystatin B (11±9%). In the majority of subjects (60 out of 65) the mean percentages of the S-modified derivatives were S-glutathionyl 53±13%, S-cysteinyl 15±5%, S-S 2-mer 32±13%. Variations of the percentages of these S-modified derivatives of cystatin B could be indicative of oral oxidative stress. As we are aware, this is the first time that S-glutathionylation and S-cysteinylation were described as extensive PTM of a salivary protein and the first time that these PTMs were detected in naturally occurring cystatin B., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Thymosin beta 10 (Tβ10) is a member of beta-thymosins (Tβs), a family of low molecular mass peptides abundant in many cell types. In previous studies, Tβs have been shown to play essential roles in many cellular functions, including cytokinesis, migration and endocytosis. Recently, Tβ10 has been found in high quantities in the saliva of human newborns, while it disappeared in the adults. On the basis of these data, it seemed of some interest to study the influence of Tβ10 during the development of the human salivary glands. To this end, we analyzed, using immunocytochemistry, the expression of Tβ10 in samples of the major and minor salivary glands obtained, at autopsy, from 2 human fetuses and 4 newborns, ranging from 13 up to 33weeks of gestation. Tβ10 immunoreactivity was detected in all salivary glands examined, with marked differences from one gland to the next, the parotid glands showing the highest Tβ10 reactivity and minor salivary glands the lowest reactivity. Marked changes were observed in Tβ10 expression and localization during embryogenesis. Tβ10 was mainly localized extracellularly in the youngest human fetuses (13weeks), in the cytoplasm of immature duct cells at 20weeks, in acinar cells and in the duct lumen in 33weeks old fetuses. Our data show, for the first time, a strong expression of Tβ10 in the human salivary glands during the initial phases of the physiological development, present at the 13th week of gestation, and suggesting a role for the peptide in the salivary glands' organogenesis., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

RP-HPLC-ESI-MS profile of saliva samples from human preterm newborn showed a protein peak in the elution range 26.6-27.6min. Deconvolution of ESI-MS spectra revealed the presence of two proteins with average molecular mass (Mav) values of 17,239+/-3Da and 18,065+/-3Da in 9 samples, with Mav value of 17,239+/-3Da in 4 samples and Mav value of 18,065+/-3Da in 2 samples. MALDI-TOF-MS analysis of tryptic digest allowed identifying the proteins as two isoforms of small proline-rich protein 3 and cDNA amplification of RNA extracts from oral mucosa, parotid and submandibular gland samples, obtained at fetal autopsy, provided two nucleotide sequences in agreement with those reported in the literature. The two proteins differ for an octapeptide repeat (GCTKVPEP) and the substitution Leu-->Val, at position 148 and 140 of the mature form of the 18,065 and 17,239Da protein, respectively. During maturation the two proteins undergo two post-translational modifications, corresponding to N-terminal acetylation and removal of the initiator methionine. cDNA amplification did not allow to clarify if the proteins found in saliva originated from cellular shedding of the epithelium and/or secretion., (Copyright 2010 Elsevier Inc. All rights reserved.)

Sjögren's syndrome (SS) is a chronic, inflammatory, autoimmune disease characterized by lymphocytic infiltration of the exocrine glands leading to qualitatively altered and diminished or absent salivary and lachrymal secretion, and by marked B-cell hyperreactivity. Many efforts have been made to define a panel of salivary and lachrymal markers helpful to design diagnostic tests able to replace blood tests and tissue biopsies for the diagnosis of primary and secondary SS. Several proteomic-based studies have indicated that a number of proteins and peptides can be considered SS biomarkers, being 2-3-fold up- or down-regulated compared to normal subject or having an exclusive presence in the saliva or tears of SS patients. Unfortunately, several factors make it difficult to define a comprehensive salivary and lachrymal panel of markers of SS, as the lack of a comprehensive proteomic analysis of human tears and saliva of healthy subjects, the lack of uniform protocols to collect and treat these samples, and the high grade of posttranslational modification of the proteins in these fluids.

Capillary electrophoresis (CE) coupled to tandem mass spectrometry was applied to the chiral separation of baclofen using sulfobutylether-beta-cyclodextrin chiral selector in partial filling counter current mode. On-line UV detection was simultaneously used. Method optimization was performed by studying the effect of cyclodextrin and BGE concentration as well as sheath liquid composition on analyte migration time and enantiomeric resolution. The cyclodextrin showed stereoselective complexation towards baclofen enantiomers, allowing chiral resolution at low concentration. The CE capillary protrusion from the ESI needle relevantly affected the chiral resolution and the analyte migration time. Complete enantiomeric separation was obtained by using 0.25 M formic acid BGE containing 1.75 mM of chiral selector and water/methanol (30:70, v/v) 3% formic acid as sheath liquid. The method exhibited a LOD of 0.1 microg/mL (racemic concentration) in MS3 product ion scan mode of detection and was applied to the analysis of racemic baclofen in pharmaceutical formulations.

Histatin 5 is a cationic salivary peptide with strong candidacidal and bactericidal activity at physiological concentration. In this paper we demonstrate by optical spectroscopy and ESI-IT-MS experiments that a synthetic peptide related to the N-terminus of histatin 5 specifically binds copper ions in vitro and that the complex metal-peptide generates reactive oxygen species at physiological concentration of ascorbate, leading to significant auto-oxidation of the peptide within short reaction time. The oxidative activity of this peptide is associated to the presence of a specific metal binding site present at its N-terminus. The motif is constituted by the amino acid sequence NH(2)-Asp-Ser-His, representing a copper and nickel amino terminal binding site, known as "ATCUN motif". The results of the study suggest that the production of reactive oxygen species can be an intrinsic property of histatin 5 connected to its ability to bind metals.

A capillary electrophoresis apparatus was used as sampler for flow injection analysis (FIA) in tandem mass spectrometry of L-carnitine and its acetyl- and propionyl-metabolites in human plasma. The capillary electrophoresis instrument was coupled to the ion trap mass spectrometer by an electrospray ionization coaxial sheath liquid interface. The electrophoresis capillary introduced the sample directly into the source by applying a prolonged sample injection. The use of the capillary electrophoresis apparatus miniaturised the FIA procedure, substantially reducing the quantities of solvents and samples used, and allowed rapid automated sequential analyses. The method was optimised and validated using a dialyzed human plasma matrix. The plasma samples were analysed after a simple, rapid deproteinisation procedure with acetonitrile and diluted 70 times before direct injection into the mass spectrometer for product ion scan MS/MS analysis in positive ionisation. The total analysis time was 5 min, including capillary preconditioning and acquisition time (3 min). The method was sensitive, allowing the determination of L-, L-acetyl- and L-propionyl-carnitines at 140, 14 and 3.6 nM concentrations (injected values) corresponding to lower limit of quantitation values in plasma of 10, 1 and 0.25 microM, respectively. The method was processed for full validation and applied to the analysis of L-carnitine and its short chain derivatives in human plasma samples.

Background: The molecular monitoring of circulating tumor cells by reverse transcriptase-PCR (RT-PCR) for patients with melanoma, is still under debate. It may be affected by: a) pre-analytical variability, b) frequency of melanoma-associated gene transcripts and c) the reliability of the methods employed. Few commercial methods are available for the detection of tyrosinase mRNA in blood., Objective: Comparison between two RT-PCR-nested methods with a third one based on real-time methodology, for detection and quantitation of tyrosinase transcripts, respectively., Methods: Sixty-two melanoma patients with different AJCC stages and 20 healthy subjects were enrolled. All blood samples were extracted in duplicate with two different methods. Two nested-PCR methods (one commercial and one in house) plus a real time commercial kit were employed., Results: The two nested PCR methods employed were overimposable, specific and sensitive, at least in the stage III, where there was a concordance between sentinel lymph nodes detection and blood tyrosinase positivity. The different extraction methods did not affect the quality of results, while the commercial real-time kit cannot be used., Conclusion: Tyrosinase mRNA detection may be therefore employed to monitor the melanoma patients over time in function of response to therapy.

We propose a method for the simultaneous determination of hippuric acid (HA) and creatinine based on capillary micellar electrokinetic chromatography. Experimental conditions were 20 mM sodium phosphate, pH 7.20, 25 mM sodium dodecyl sulfate, 5% (v/v) acetonitrile. Electropherograms evidenced HA and creatinine peaks in less than 12 min. The method showed good linearity for both analytes and satisfactory within-day precision. The present method, which is accurate, sensitive, rapid and simple, may be applied to single-spot urine samples.

In the present study we analysed the effects of S-nitrosocysteine (CysNO) on adult human red blood cell metabolism and observed that metabolic response depended on the degree of cell oxygenation. In particular, glucose metabolised through the pentose phosphate pathway (PPP) was higher in treated erythrocytes than in untreated cells only at high O(2) pressure. Since, following the treatment of intact cells with CysNO, glucose-6-phosphate dehydrogenase (G6PD) and phosphofructokinase (PFK) activities did not evidence any significant alteration, the possibility that the stimulation of PPP was triggered by a CysNO mediated modification of these enzymes was excluded. Intracellular S-nitrosoglutathione (GSNO), detected only in treated red blood cells, may be linked solely to the exposition to the NO donor. A possible rationalisation of the different metabolic behaviour shown by erythrocytes as a function of their oxygenation state is proposed. It takes into account the different route of catabolic degradation observed in vitro for GSNO under aerobic and anaerobic condition., ((c) 2002 Elsevier Science (USA).)